African swine fever virus pMGF360-3 L suppresses innate immunity by facilitating CMA-dependent SKP1 degradation to promote BNIP3-mediated mitophagy

Abstract

African swine fever (ASF) is an acute, hemorrhagic, and highly contagious disease caused by African swine fever virus (ASFV), which causes severe economic losses in the swine industry. ASFV has evolved multiple strategies to evade host antiviral immune responses. Here, we report that ASFV pMGF360-3 L promotes host mitophagy by manipulating chaperone-mediated autophagy (CMA), thereby inhibiting the production of type I interferon (IFNB/IFN-β). Mechanistically, pMGF360-3 L targets the SKP1 protein via its N-terminal ankyrin (ANK) repeat domain, promoting the degradation of SKP1 through the CMA pathway, which inhibits the proteasomal degradation of BNIP3 to increase its expression level in mitochondria. Subsequently, BNIP3 binds to MAP1LC3B/LC3B to induce mitophagy, a process that leads to the degradation of mitochondria. Notably, the CMA-mediated degradation of SKP1 depends on its K94 site, and the SKP1-BNIP3 axis is critical for pMGF360-3 L-mediated IFNB inhibition. In summary, our study reveals a mechanism through which ASFV pMGF360-3 L facilities CMA-dependent degradation of the E3 complex component SKP1. This stabilizes mitochondrial BNIP3 to initiate mitophagy and block IFNB production. This deepens our understanding of the immune evasion strategies of ASFV and provides potential drug targets for controlling viral infection.Abbreviations: 3-MA: 3-methyladenine; ASFV: African swine fever virus; BafA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; CMA: chaperone-mediated autophagy; co-IP: co-immunoprecipitation; CQ: chloroquine; CHX: cycloheximide; CUL1: cullin 1; DAPI: 4’, 6-diamidino-2’-phenylindole; EV: emptor vector; FBXL4: F-box and leucine rich repeat protein 4; hpi: hours post-infection; IFNB: interferon beta; ISGs: IFN-stimulated genes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MG132: cbz-leu-leu-leucinal; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMs: porcine alveolar macrophages; PPTC7: protein phosphatase targeting COQ7; RBX1: ring-box 1; RT-PCR: real-time polymerase chain reaction; siRNA: small interfering RNA; SKP1: S-phase kinase associated protein 1; TCID50: 50% tissue culture infectious doses; Ub: ubiquitin; WCL: whole-cell lysate; WT: wild-type.